The present invention relates to plant molecular biology. In particular, it relates to promoter sequences useful for gene expression in selected plant organs and tissues.
Isolated plant promoters are useful in the genetic engineering of plants to produce transgenic plants with desired phenotypes. To produce such transgenic plants, an isolated plant promoter is inserted into a vector and operably linked to a heterologous DNA sequence. Plant cells are then transformed with the vector such that expression of the heterologous DNA is controlled by the promoter.
Some plant promoters are tissue-specific, while others are constitutive and drive expression in essentially all tissues and organs. Tissue-specific promoters can be identified from genes that are expressed in particular tissues or at particular times during development.
A need exists for a variety of different promoters to be used in the genetic engineering of plants. New tissue-specific promoters are particularly useful for the controlled expression of various nucleic acid sequences in transgenic plants. The present invention addresses these and other needs.
The present invention provides embryo specific maize metallothionein promoters. The promoters can be used to provide embryo specific expression of the heterologous sequences in plants. In particular, the promoters are useful in expression in maize. More specifically, the invention provides an isolated DNA molecule comprising base pairs 50 to 1649 of SEQ ID NO:5, or a fragment, genetic variant or deletion of such a sequence which retains the ability of functioning as an embryo specific promoter in plant cells.
The invention also provides expression cassettes comprising an embryo specific promoter operably linked to a heterologous nucleic acid sequence, wherein the promoter selectively hybridizes to a 20 consecutive base pair portion of the sequence set forth in SEQ ID NO:5.
The invention also provides an expression cassette of wherein the promoter comprises a sequence extending from about nucleotide 50 to nucleotide 1649 of SEQ ID NO:5.
The invention also provides a method of expressing a heterologous nucleic acid sequence in a plant comprising:
a) introducing into plant tissue a vector comprising an embryo specific maize metallothionein promoter operably linked to the heterologous nucleic acid sequence; and
b) regenerating the plant tissue into a whole plant.
The invention also provides an isolated DNA molecule having a 20 base pair nucleotide portion identical in sequence to a 20 consecutive base pair portion of the sequence set forth in SEQ ID NO:5.
The invention also provides transgenic plant comprising the expression cassettes described above. The plant may be any agronomically useful plant.
The term xe2x80x9cnucleic acidsxe2x80x9d, as used herein, refers to either DNA or RNA. xe2x80x9cNucleic acid sequencexe2x80x9d or xe2x80x9cpolynucleotide sequencexe2x80x9d refers to a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5xe2x80x2 to the 3xe2x80x2 end. Sequence regions on a DNA strand that are 5xe2x80x2 to the 5xe2x80x2 end of an RNA transcript encoded by the DNA are referred to as xe2x80x9cupstream sequencesxe2x80x9d. Upstream sequences are usually counted in a negative direction from the transcription start site. Sequence regions on the DNA strand that are 3xe2x80x2 to the 3xe2x80x2 end of the RNA transcript are referred to as xe2x80x9cdownstream sequences.xe2x80x9d
The term xe2x80x9cpromoterxe2x80x9d refers to a region of DNA upstream from the translational start codon and which is involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A xe2x80x9cplant promoterxe2x80x9d is a promoter capable of initiating transcription in plant cells. The term maize metallothionein promoter as used herein refers to plant promoters comprising sequences derived from the promoter region of a maize metallothlonein gene. The promoters of the invention contain tissue specific elements that allow embryo specific transcription of operably linked DNA sequences. The promoters are considered to be embryo specific promoters because transcription of the operably linked DNA is higher in embryo tissues than it is in other tissues.
A xe2x80x9ctissue-specificxe2x80x9d promoter as used herein refers to a promoter that drives expression of an operably linked nucleic acid sequence in a particular tissue in a plant or at a particular stage in the plant life-cycle.
The term xe2x80x9coperably linkedxe2x80x9d as used herein refers to linkage of a promoter upstream from a DNA sequence such that the promoter mediates transcription of the DNA sequence. It is understood that the promoter sequence aim includes transcribed sequences between the transcriptional start and the translational start and the translational start codon.
The phrase xe2x80x9cexpression cassettexe2x80x9d, refers to nucleotide sequences which are capable of affecting expression of a structural gene in hosts compatible wanh such sequences. Such cassettes include at least promoters and optionally, transcription termination signals. Additional factors necessary or helpful in effecting expression may also be used as described herein.
A xe2x80x9cheterologousxe2x80x9d nucleic acid or protein is one that originates from a foreign source (or species) or, if from the same source is modified from its original form. Thus, a heterologous promoter sequence in an expression cassette is from a source different from the source of the coding sequence, or, if from the same source, is modified from its original form. Modification may occur, e.g., by treating the DNA with a restriction enzyme to generate a promoter element that is capable of conferring tissue-specific expression on the expression cassette which includes it.
The phrases xe2x80x9cisolatedxe2x80x9d or xe2x80x9csubstantially purexe2x80x9d when referring to a polynucleotide or protein, means a chemical composition which is free of other subcellular components of the organism from which it its derived. Typically, a compound is substantially pure when at least about 85% or more of a sample exhibits a single polypeptide backbone, or polynucleotide sequence. Minor variants or chemical modifications may typically share the same polypeptide sequence. Depending on the purification procedure, purity of 85%, and preferably over 95% pure are possible. Nucleic acid and protein purity or homogeneity may be indicated by a number of means well known in the art, such as gel electrophoresis and the like.
The term xe2x80x9cplantxe2x80x9d includes whole plants, plant organs (e.g., leaves, stems roots, etc.), seeds and plant cells and progeny of same. The class of plants which can be used in the method of the invention is generally as broad as the class of higher plants amenable to transformation techniques, including both monocotyledonous and dicotyledonous plants.
xe2x80x9cPercentage of sequence identityxe2x80x9d for polynucleotides and polypeptides is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Optimal alignment of sequences for comparison may be conducted by computerized implementations of known algorithms (e.g., GAP, BESTFIT, PASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., or BlastN and BlastX available from the National Center for Biotechnology Information), or by inspection. Sequences are typically compared using GESTFIT or BlastN with default parameters.
Substantial identity of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 75% sequence identity, preferably at least 80%, more preferably at least 90% and most preferably at least 95%. Typically, two polypeptides are considered to be substantially identical if at least 40%, preferably at least 60%, more preferably at least 90%, and most preferably at least 95% are identical or conservative substitutions. Polypeptides which are xe2x80x9csubstantially similarxe2x80x9d share sequences as noted above except that residue positions which are not identical may differ by conservative amino acid changes. Conservative amino acid substitutions refer to the interchangeability of residues having simnilar side chains.
Another indication that polynucleotide sequences are substantially identical is if two molecules selectively hybridize to each other under stringent conditions. The phrase xe2x80x9cselectively hybridizing toxe2x80x9d refers to a nucleic acid probe that hybridizes or binds only to a particular target DNA or RNA sequence when the target sequences are present in a preparation of total cellular DNA or RNA. Stringent conditions are sequence dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 20xc2x0 C. lower than the thermal melting point (Tm) for the specific sequence at a define ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
SEQ ID NO:1 is the DNA sequence of the SK primer.
SEQ ID NO:2 is cDNA sequence for Maize Ec Metallothionein
SEQ ID NO:3 is the DNA sequence for clone 651, containing the first 233 bases of the coding region for Maize Ec Metallothionein.
SEQ ID NO:4 is the DNA sequence for the T3 primer.
SEQ ID NO:5 is the DNA sequence for clone MGN 111-1.
The present invention provides new plant promoter sequences useful for expression of desired nucleic acid sequences in plant embryos. In particular, the invention provides isolated nucleic acid molecules comprising sequences from promoters derived from a maize metallothionein gene. The promoter sequences of the invention can be used to drive expression of a variety of heterologous nucleic acids sequences in embryo tissue of transgenic plants.
I. Isolation of Maize Metallothionein Promoters
The promoter sequences of the invention are typically identical to or show substantial sequence identity (determined as described above) to portions of the maize metallothionein promoter nucleotide sequence depicted in bp 50-1649 of SEQ ID NO: 5. A number of different promoters having homology or substantial sequence identity to the promoter sequences of SEQ ID NO:5 can be isolated from maize.
Maize metallothionein promoter sequences typically hybridize to a nucleic acid having a sequence as shown in bp 50-1649 of SEQ ID NO: 5 under stringent conditions. Typically stringent conditions for a Southern blot protocol involve washing at 55xc2x0 C. with 0.2xc3x97SSC.
There are a variety of methods that may be used for isolation of maize metallothionein promoter sequences. For example, DNA can be isolated from a genomic library using labeled nucleic acid probes having sequences complementary to the sequences disclosed here. Full-length probes may be used, or oligonucleotide probes may also be generated. Alternatively, genomic clones comprising the genes can be isolated and the 5xe2x80x2 end of the clones can be subcloned to provide the promoter sequences. Techniques for nucleic acid manipulation of genes such as subcloning nucleic acid sequences encoding polypeptides into expression vectors, labeling probes, DNA hybridization, and the like are described generally in Sambrook, et al., Molecular Cloningxe2x80x94A Laboratory Manual (2nd Ed.), Vol. 103, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989). This manual is hereinafter referred to as xe2x80x9cSambrook, et al.xe2x80x9d
In addition to screening using the sequences disclosed here, techniques designed to identify sequences specific to a particular tissue or cell types can be used to isolate sequences of the invention (see, e.g., Sambrook, et al.) Such techniques include differential hybridization techniques as described in the example section or in Gurr, et al. Mol. Gen. Genet. 226:361-366 (1991). In addition, subtractive hybridization techniques can be used to prepare specific probes for screening cbNA or genomic libraries. These techniques can also be used to prepare subtracted libraries enriched for the desired sequences. Once a desired genomic clone is identified, the 5xe2x80x2 sequences can be analyzed to identify the promoter sequence from the gene. This can be accomplished by inserting 5xe2x80x2 sequences in front of a promoterless reporter gene (e.g., GUS) to identify those regions which can drive expression of a structural gene.
Nucleic acid amplification techniques such as polymerase chain reaction (PCR) technology, can also be used to amplify the desired genes and promoter sequences from mRNA, from cDNA, and from genomic libraries or cDNA libraries. In PCR techniques, oligonucleotide primers based on the sequences disclosed here and complementary to the two 5xe2x80x2 and 3xe2x80x2 borders of the DNA region to be amplified are synthesized. The polymerase chain reaction is then carried out using the two primers. See PCR Protocols: A Guide to Methods and Applications (Innis, M, Gelfand, D., Sncnsky, J. and White, T., eds.), Academic Press, San Diego (1990). Primer s can be selected to amplify the entire regions encoding a full-length isocitrate lyase or its promoter. PCR can also be used to amplify smaller DNA segments of these regions as desired.
Oligonucleotides for use as primer or probes in the above-mentioned procedures can be chemically synthesized according to standard techniques such as the solid phase phosphoramidite triester method first described by Beaucage, et al. Tetrahedron Lett. 22(20): 1859-1862 (1981), using an automated synthesizer, as described in Needham-Van Devanter, et al., Nucleic Acids Res. 12:6159-6168 (1984).
Typically, the maize metallothionein promoters of the invention will be about 170 nucleotides to about 1800 nucleotides in length, usually between about 200 to about 1500 nucleotides.
As demonstrated below, sequences which confer tissue specific expression are found in the promoters of the invention. Thus, heterologous promoters can be constructed which have tissue specific expression as a result of the presence of tissue specific elements contained in these sequences.
An additional important element is the 5xe2x80x2 untranslated leader, i.e. the 5xe2x80x2 end of the mRNA extending from the 5xe2x80x2 CAP site to the AUG translation initiation codon of the mRNA. The leader plays a critical role in translation initiation and in regulation of gene expression. For most eukaryotic mRNAs, translation initiates with the binding of the CAP binding protein to the mRNA CAP. This is then followed by the binding of several other translation factors, as well as the 43S ribosome pre-initiation complex. This complex travels down the mRNA molecule while scanning for an AUG initiation codon in an appropriate sequence context. Once this has been found, and with the addition of the 60S ribosomal subunit, the complete 80S initiation complex initiates protein translation. Pain (1986); Moldave (1985); Kozak (1986). Optimization of the leader sequence for binding to the ribosome complex has been shown to increase gene expression as a direct result of improved translation initiation efficiency. Significant increases in gene expression have been produced by addition of leader sequences from plant viruses or heat shock genes. Raju et al. (1993); (Austin, 1994). Dietrich et al. (1987) reported that the length of the 5xe2x80x2 non-translated leader was important for gene expression in protoplasts.
Specific embodiments of the invention include the following portions of SEQ ID NO:5:
II. Construction of Expression Cassettes and Vectors
The methods required for construction of vectors containing expression cassettes comprising a promoter of the invention operably linked to desired sequence are well known. The minimal requirements of the vector are that the desired nucleic acid sequence be introduced in a relatively intact state. Thus, any vector which will produce a plant carrying the introduced DNA sequence should be sufficient. The selection of vectors and methods to construct them are commonly known to persons of ordinary skill in the art and are described in general technical references (See, in general, Methods in Enzymology Vol. 153 (xe2x80x9cRecombinant DNA Part Dxe2x80x9d) 1987, Wu and Grossman Eds., Academic Press.
The recombinant vectors of the present invention typically comprise an expression cassette designed for initiating transcription of the desired polynucleotide sequences in plants. Companion sequences, of bacterial origin, are also included to allow the vector to be cloned in a bacterial host. The vector will preferably contain, a broad host range prokaryote origin of replication. A selectable marker should also be included to allow selection of bacterial cells bearing the desired construct. Suitable prokaryotic selectable markers include resistance to antibiotics such as kanamycin or tetracycline.
For expression of polypeptides in plants, the recombinant expression cassette will contain, in addition to the desired polynucleotide sequence and the promoter sequence of the invention, a translation initiation site (if the sequence to be transcribed lacks one), and a transcription termination sequence. Unique restriction enzyme sites at the 5xe2x80x2 and 3xe2x80x2 ends of the cassette are typically included to allow for easy insertion into preexisting vector.
In the construction of heterologous promoter/structural gene combinations, the promoter is preferably postponed about the same distance from the heterologous transcription start site as it is from the transcription start site in the natural setting. As is known in the art, however, some variations in this can be accommodated without loss of promoter function.
As noted above, an expression cassette should also contain a transcription termination region downstream of the structural gene to provide for efficient termination. The termination region may be obtained from the same gene as the promoter sequence or may be obtained from different genes.
If the mRNA encoded by the structural gene is to be efficiently translated, polyadenylation sequences are also commonly added to the vector construct. Alber and Kawasaki, Mo. and Appl. Genet, 1:419-434, 1982, Polyadenylation sequences include, but are not limited to the Agrobacterium octopine synthase signal (Gielen et al., EMBO J., 3:835-846, 1984) or the nopaline synthase signal (Depicker et al.) Mol. and Appi. Genet, 1:561-573,1982).
The vector will also typically contain a selectable marker gene by which transformed plant cells can be identified in culture. Usually, the marker gene will encode antibiotic or herbicide resistance. These markers include resistance to G418, hygromycin, bleomycin, kanamycin, gentamicin, Basta(trademark), and chlorsulfuron. After transforming the plant cells, those cells having the vector will be identified by their ability to grow in a medium containing the particular antibiotic.
Examples of suitable structural genes that can be expressed using the promoter sequences of the invention include genes for herbicide resistance; genes for fungal disease resistance (e.g., chitinases and glucanases); genes for bacterial disease resistance (e.g., cecropins); genes for insect resistance (e.g., B thuringiensis toxin); and genes which modify the oil or amino acid contents of the embryo.
III. Production of Transgenic Plants
Techniques for transforming a wide variety of higher plant species are well known and described in the literature. See, for example, Weising, et al., Ann. Rev. Genet. 22:421-477 (1988). DNA constructs containing the promoter sequenced linked to heterologous DNA can be introduced into genome of the desired plant host by a variety of conventional techniques. For example, the DNA construct may be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation and microinjection of plant cell protoplasts. Alternatively, the DNA constructs can be introduced directly to plant tissue using ballistic methods, such as DNA particle bombardment. Usually, the DNA constructs are combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector.
Direct transformation techniques are known in the art and well described in the scientific literature. The introduction of DNA constructs using polyethylene glycol precipitation is described in Paszkowski, et al., Embo J. 3:2717-2722 (1984). Electroporation techniques are described in Fromm, et al., Proc. Natl. Acad. Sci. USA 82:5824 (1985). Ballistic transformation techniques are described in Klein, et al., Nature 327:70-73 (1987). Whisker-mediated transformation is described in Frame et al., Plant J. 6:941-948 (1994).
Agrobacterium-mediated transformation techniques are the most commonly used techniques and are well described in the scientific literature. See, for example Horsch, et al Science 233:496498 (1984), and Fraley, et al., Proc. Natl. Acad. Sci. USA 80:4803 (1983).
The expression of the heterologous DNA sequences can be detected in a variety of ways, depending on the nature of heterologous sequences. For instance, resistance to an herbicide or pathogen can be detected by treatment with the herbicide or pathogen. Expression can be detected by measurement of the specific RNA transcription product. This can be done by, for example, Northern blot procedures. If heterologous DNA sequences encode a novel protein, the protein product may be assayed, for instance, by its function or by a variety of immunoassay techniques.
Transformed plant cells which are derived by any of the above transformation techniques can be cultured to regenerate a whole plant which possesses for desired. transformed phenotype. Plant regeneration from cultured protoplasts is described in Evans, et al., Protoplast Isolation and Culture, Handbook of Plant Cell Culture, MacMillan Publishing Company, New York, pp. 124-176 (1983); and Binding, Regeneration of Plants, Plant Protoplasts, CRC Press, Boca Raton, pp. 21-73 (1985). Regeneration can also be obtained from plant callus, explants, organs, or part thereof. Such regeneration techniques are described generally in Klee, et al., Ann Rev. of Plant Phys. 38:467-486 (1987)l
One of skill will recognize that, after an expression cassette comprising a maize metallothionein promoter sequence is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
The promoter sequences of the invention can be used in the transformation of any plant, including both dicots and monocots. Transformation of dicots is described in references above. Transformation of monocots is known using various techniques including electroporation (e.g., Shimamoto, et al., Nature 338:274-476 (1992); ballistics (e.g., European Patent Application 270,356); and Agrobacterium (e.g., Bytebier, et al., Proc. Natl. Acad. Sci USA 84:5345-5349 (1987).
The methods and compositions of the invention have use over a broad range of types of plants.